Difference between revisions of "Part:BBa K594002:Design"
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Besides, when we were looking for leuB gene, we also try to find its RBS, but unluckily, we didn't find its RBS on NCBI, its location is closely linked to leuA's location. | Besides, when we were looking for leuB gene, we also try to find its RBS, but unluckily, we didn't find its RBS on NCBI, its location is closely linked to leuA's location. | ||
| + | ===The verification=== | ||
| + | We have sent the sinI to the sequencing center, and it proves that it is 100% correct. | ||
| + | the sequecing result, | ||
| + | |||
| + | AAATCTTGACCTTAACCTATAAAAATAGGCGTATCACGAGGCAGAATTTCAGATAAAAAAAATCCTTAGCTTTCGCTAAGGATGA<br> | ||
| + | TTTCTGGAATTCGCGGCCGCTTCTAGATGTCGAAGAATTACCATATTGCCGTATTGCCGGGGGACGGTATTGGTCCGGAAGTGAT<br> | ||
| + | GACCCAGGCGCTGAAAGTGCTGGATGCCGTGCGCAACCGCTTTGCGATGCGCATCACCACCAGCCATTACGATGTAGGCGGCGCA<br> | ||
| + | GCCATTGATAACCACGGGCAACCACTGCCGCCTGCGACGGTTGAAGGTTGTGAGCAAGCCGATGCCGTGCTGTTTGGCTCGGTAG<br> | ||
| + | GCGGCCCGAAGTGGGAACATTTACCACCAGACCAGCAACCAGAACGCGGCGCGCTGCTGCCTCTGCGTAAGCACTTCAAATTATT<br> | ||
| + | CAGCAACCTGCGCCCGGCAAAACTGTATCAGGGGCTGGAAGCATTCTGTCCGCTGCGTGCAGACATTGCCGCAAACGGCTTCGAC<br> | ||
| + | ATCCTGTGTGTGCGCGAACTGACCGGCGGCATCTATTTCGGTCAGCCAAAAGGCCGCGAAGGTAGCGGACAATATGAAAAAGCCT<br> | ||
| + | TTGATACCGAGGTGTATCACCGTTTTGAGATCGAACGTATCGCCCGCATCGCGTTTGAATCTGCTCGCAAGCGTCGCCACAAAGT<br> | ||
| + | GACGTCGATCGATAAAGCCAACGTGCTGCAATCCTCTATTTTATGGCGGGAGATCGTTAACGAGATCGCCACGGAATACCCGGAT<br> | ||
| + | GTCGAACTGGCGCATATGTACATCGACAACGCCACCATGCAGCTGATTAAAGATCCATCACAGTTTGACGTTCTGCTGTGCTCCA<br> | ||
| + | ACCTGTTTGGCGACATTCTGTCTGACGAGTGCGCAATGATCACTGGCTCGATGGGGATGTTGCCTTCCGCCAGCCTGAACGAGCA<br> | ||
| + | AGGTTTTGGACTGTATGAACCGGCGGGCGGCTCGGCACCAGATATCGCAGGCAAAAACATCGCCAACCCGATTGCACAAATCCTT<br> | ||
| + | TCGCTGGCACTGCTGCTGCGTTACAGCCTGGATGCCGATGATGCGGCTTGCGCCATTGAACGCGCCATTAACCGCGCATTAGAAG<br> | ||
| + | AAGGCATTCGCACCGGGGATTTAGCCCGTGGCGCTGCCGCCGTTAGTACCGATGAAATGGGCGATATCATTGCCCGCTATGTAGC<br> | ||
| + | AGAAGGGGTGTAATACTAGTAGCGGCCGCTGCAGTCCGGCAAAAAAGGGCAAGGTGTCACCACCCTGCCCTTTTTCTTTAAAACC<br> | ||
| + | GAAAAGATTACTTCGCGTTATGCAGGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTCCGCTGCGCAGCCCCCG | ||
| + | |||
| + | |||
| + | We use NCBI blast to analysis the sequence, finally get the result. | ||
| + | |||
| + | Score = 2017 bits (1092), Expect = 0.0, Identities = 1092/1092 (100%), Gaps = 0/1092 (0%) | ||
| + | Strand=Plus/Plus | ||
| + | |||
| + | So the leuB is right, when added proper promoter and RBS, it will work, | ||
===Source=== | ===Source=== | ||
Revision as of 03:22, 6 October 2011
subunit of a 3-isopropylmalate dehydrogenase from E.coli BL21
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
Design Notes
Besides, when we were looking for leuB gene, we also try to find its RBS, but unluckily, we didn't find its RBS on NCBI, its location is closely linked to leuA's location.
The verification
We have sent the sinI to the sequencing center, and it proves that it is 100% correct.
the sequecing result,
AAATCTTGACCTTAACCTATAAAAATAGGCGTATCACGAGGCAGAATTTCAGATAAAAAAAATCCTTAGCTTTCGCTAAGGATGA
TTTCTGGAATTCGCGGCCGCTTCTAGATGTCGAAGAATTACCATATTGCCGTATTGCCGGGGGACGGTATTGGTCCGGAAGTGAT
GACCCAGGCGCTGAAAGTGCTGGATGCCGTGCGCAACCGCTTTGCGATGCGCATCACCACCAGCCATTACGATGTAGGCGGCGCA
GCCATTGATAACCACGGGCAACCACTGCCGCCTGCGACGGTTGAAGGTTGTGAGCAAGCCGATGCCGTGCTGTTTGGCTCGGTAG
GCGGCCCGAAGTGGGAACATTTACCACCAGACCAGCAACCAGAACGCGGCGCGCTGCTGCCTCTGCGTAAGCACTTCAAATTATT
CAGCAACCTGCGCCCGGCAAAACTGTATCAGGGGCTGGAAGCATTCTGTCCGCTGCGTGCAGACATTGCCGCAAACGGCTTCGAC
ATCCTGTGTGTGCGCGAACTGACCGGCGGCATCTATTTCGGTCAGCCAAAAGGCCGCGAAGGTAGCGGACAATATGAAAAAGCCT
TTGATACCGAGGTGTATCACCGTTTTGAGATCGAACGTATCGCCCGCATCGCGTTTGAATCTGCTCGCAAGCGTCGCCACAAAGT
GACGTCGATCGATAAAGCCAACGTGCTGCAATCCTCTATTTTATGGCGGGAGATCGTTAACGAGATCGCCACGGAATACCCGGAT
GTCGAACTGGCGCATATGTACATCGACAACGCCACCATGCAGCTGATTAAAGATCCATCACAGTTTGACGTTCTGCTGTGCTCCA
ACCTGTTTGGCGACATTCTGTCTGACGAGTGCGCAATGATCACTGGCTCGATGGGGATGTTGCCTTCCGCCAGCCTGAACGAGCA
AGGTTTTGGACTGTATGAACCGGCGGGCGGCTCGGCACCAGATATCGCAGGCAAAAACATCGCCAACCCGATTGCACAAATCCTT
TCGCTGGCACTGCTGCTGCGTTACAGCCTGGATGCCGATGATGCGGCTTGCGCCATTGAACGCGCCATTAACCGCGCATTAGAAG
AAGGCATTCGCACCGGGGATTTAGCCCGTGGCGCTGCCGCCGTTAGTACCGATGAAATGGGCGATATCATTGCCCGCTATGTAGC
AGAAGGGGTGTAATACTAGTAGCGGCCGCTGCAGTCCGGCAAAAAAGGGCAAGGTGTCACCACCCTGCCCTTTTTCTTTAAAACC
GAAAAGATTACTTCGCGTTATGCAGGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTCCGCTGCGCAGCCCCCG
We use NCBI blast to analysis the sequence, finally get the result.
Score = 2017 bits (1092), Expect = 0.0, Identities = 1092/1092 (100%), Gaps = 0/1092 (0%) Strand=Plus/Plus
So the leuB is right, when added proper promoter and RBS, it will work,
Source
This leuB is from the leu operon in BL 21(E.coli),we get BL21 from Takara and found its sequence in NCBI, then cloned it with a Pfu PCR.
References
1.J. M. Somers, A. Amzallag1, and R. B. Middleton2，Genetic Fine Structure of the Leucine Operon of Escherichia coli K-12，J Bacteriol. 1973 March; 113(3): 1268-1272
2.Wallon G, Yamamoto K, Kirino H, Yamagishi A, Lovett ST, Petsko GA, Oshima T (1997). "Purification, catalytic properties and thermostability of 3-isopropylmalate dehydrogenase from Escherichia coli." Biochim Biophys Acta 1337(1);105-12. PMID: 9003442
3. Wallon G, Kryger G, Lovett ST, Oshima T, Ringe D, Petsko GA (1997). "Crystal structures of Escherichia coli and Salmonella typhimurium 3-isopropylmalate dehydrogenase and comparison with their thermophilic counterpart from Thermus thermophilus." J Mol Biol 266(5);1016-31. PMID: 9086278
4.Wright BE, Longacre A, Reimers JM (1999). "Hypermutation in derepressed operons of Escherichia coli K12." Proc Natl Acad Sci U S A 96(9);5089-94. PMID: 10220423
5. Yang HL, Kessler DP (1974). "Genetic analysis of the leucine region in Escherichia coli B-r: gene-enzyme assignments." J Bacteriol 117(1);63-72. PMID: 4587614